Towards the Enhancement of Essential Oil Components' Antimicrobial Activity Using New Zein Protein-Gated Mesoporous Silica Microdevices.

The development of new food preservatives is essential to prevent foodborne outbreaks or food spoilage due to microbial growth, enzymatic activity or oxidation. Furthermore, new compounds that substitute the commonly used synthetic food preservatives are needed to stifle the rising problem of microbial resistance. In this scenario, we report herein, as far as we know, for the first time the use of the zein protein as a gating moiety and its application for the controlled release of essential oil components (EOCs). The design of microdevices consist of mesoporous silica particles loaded with essential oils components (thymol, carvacrol and cinnamaldehyde) and functionalized with the zein (prolamin) protein found in corn as a molecular gate. The zein protein grafted on the synthesized microdevices is degraded by the proteolytic action of bacterial enzymatic secretions with the consequent release of the loaded essential oil components efficiently inhibiting bacterial growth. The results allow us to conclude that the new microdevice presented here loaded with the essential oil component cinnamaldehyde improved the antimicrobial properties of the free compound by decreasing volatility and increasing local concentration.

In vitro antimicrobial activity of immobilised essential oil components against Helicobacter pylori.

Available disinfection methods and therapies against Helicobacter pylori have multiple disadvantages, such as increased prevalence of antibiotic-resistant strains, which requires the search for novel effective antimicrobial agents against H. pylori. Among them, naturally-occurring antimicrobial compounds, like essential oil components (EOCs), have been reported as substances with anti-H. pylori potential. To avoid the disadvantages associated with using EOCs in their free form, including volatility, low water solubility and intense sensory properties, their immobilisation in inert supports has recently been developed. This study sought to evaluate the inhibitory properties of EOCs immobilised on silica microparticles against H. pylori and to elucidate the mechanism of action of the immobilised antimicrobials. After the preparation and characterisation of the antimicrobial supports, the susceptibility of H. pylori in the presence of the immobilised compounds was assessed by plate count, fluorescent viability staining and direct viable count-fluorescent in situ hybridisation analyses. The antimicrobial supports were found to inhibit H. pylori growth, and to induce morphological and metabolic alterations to the H. pylori membrane, with a minimum bactericidal concentration value between 25 and 50 µg/ml according to the tested EOC. These findings indicate that immobilised EOCs can be used as potential antimicrobial agents for H. pylori clearance and treatment.

Remarkable enhancement of cinnamaldehyde antimicrobial activity encapsulated in capped mesoporous nanoparticles: A new "nanokiller" approach in the era of antimicrobial resistance.

Combating antimicrobial resistance is one of the biggest health challenges because of the ineffectiveness of standard biocide treatments. This challenge could be approached using natural products, which have demonstrated powerful therapeutics against multidrug-resistant microbes. In the present work, a nanodevice consisting of mesoporous silica nanoparticles loaded with an essential oil component (cinnamaldehyde) and functionalized with the polypeptide ε-poly-l-lysine is developed and used as an antimicrobial agent. In the presence of the corresponding stimuli (i.e., exogenous proteolytic enzymes from bacteria or fungi), the polypeptide is hydrolyzed, and the cinnamaldehyde delivery is enhanced. The nanodevice's release mechanism and efficacy are evaluated in vitro against the pathogenic microorganisms Escherichia coli, Staphylococcus aureus, and Candida albicans. The results demonstrate that the new device increases the delivery of the cinnamaldehyde via a biocontrolled uncapping mechanism triggered by proteolytic enzymes. Moreover, the nanodevice notably improves the antimicrobial efficacy of cinnamaldehyde when compared to the free compound, ca. 52-fold for E. coli, ca. 60-fold for S. aureus, and ca. 7-fold for C. albicans. The enhancement of the antimicrobial activity of the essential oil component is attributed to the decrease of its volatility due to its encapsulation in the porous silica matrix and the increase of its local concentration when released due to the presence of microorganisms.

Impact of food preservatives based on immobilized phenolic compounds on an in vitro model of human gut microbiota.

To address concerns about the biocompatibility of novel phenolic immobilization-based food preservatives, their impact on the composition and metabonomic profile of a defined community of human gut microbiota was evaluated. Three phenolics (eugenol, vanillin and ferulic acid) presented in two forms (free or immobilized on different supports) were tested at two concentration levels (0.5 and 2 mg/mL). Free eugenol was the phenolic with the greatest impact on gut microbiota, with a remarkable increase in the abundance of Lachnospiraceae and Akkermansiaceae families. In contrast, immobilized phenolics produced an increase in the abundance of Bacteroides with a reduction in the ratio of Firmicutes to Bacteroidetes. The metabonomic profile was also affected by free and immobilized phenolics differently in terms of fermentation by-products and phenolic biotransformation metabolites. Thus the results suggest the importance of evaluating the impact of new compounds or materials added to food on human gut microbiota and their potential use to modulate microbiota composition.

Immobilization of Natural Antimicrobial Compounds on Food-Grade Supports as a New Strategy to Preserve Fruit-Derived Foods.

The use of natural antimicrobials in the food industry is being proposed as an eco-friendly postharvest technology to preserve fruit-derived foods. In this context, this systematic review aims to describe and discuss the application of naturally occurring antimicrobial compounds in the processing of fruit-derived foods by the PRISMA methodology. In a first step, the use of free natural antimicrobials was investigated as an approach to identify the main families of bioactive compounds employed as food preservatives and the current limitations of this dosage form. Then, the use of immobilized antimicrobials, in an innovative dosage form, was studied by distinguishing two main applications: addition to the food matrix as preservatives or use during processing as technological aids. Having identified the different examples of the immobilization of natural antimicrobial compounds on food-grade supports, the mechanisms of immobilization were studied in detail to provide synthesis and characterization guidelines for future developments. Finally, the contribution of this new technology to decarbonization and energy efficiency of the fruit-derived processing sector and circular economy is discussed in this review.

In vitro susceptibility of human gut microbes to potential food preservatives based on immobilized phenolic compounds.

The development of novel food preservatives based on natural antimicrobials such as phenolic compounds is increasing, but their safety should be established before use, including evaluating their impact on the gut microbiota. This work explored the influence of antimicrobial phenolics presented in different forms on selected human gut microbiota members through in vitro susceptibility tests. The bacteria tested exhibited a wide range of susceptibilities to phenolics depending on the molecule structure and mode of administration. Agathobacter rectalis and Clostridium spiroforme, members of the phylum Firmicutes, were the most sensitive strains. Susceptibility was strain- and species-specific, suggesting that it may not be possible to easily extrapolate results across the human microbiome in general. Species of other phyla including Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia were more resistant than Firmicutes, with growth of some strains even enhanced. Our results provide insights into the biocompatibility of free and immobilized phenolics as potential food additives.

Influence of free and immobilized chitosan on a defined human gut microbial ecosystem.

In this work, the influence of different forms of presentation of chitosan in the human gut microbiota with a defined bacterial community was evaluated. First, the susceptibility of individual gut bacterial isolates against chitosan was studied within a concentration range between 0.125 and 1 mg/mL. Then, the impact of chitosan (0.25 and 1 mg/mL) on a defined human gut microbial ecosystem was studied by metagenomic and metabonomic analyses. The results showed that chitosan in its free form had a high impact on individual isolates with a minimum inhibitory concentration below 1 mg/mL for most of the strains studied. In comparison, chitosan immobilized in the different carriers displayed a diverse effect on gut microbiota. The most susceptible strains were Agathobacter rectalis strain 16-6-I 1 FAA, Clostridium spiroforme strain 16-6-I 21 FAA and Mediterraneibacter faecis strain 16-6-I 30 FAA. The impact of the different modes of presentation of chitosan was strain-specific and species-specific when compared to results obtained from analysis of other strains within the genera Agathobacter, Clostridium and Mediterraneibacter, and therefore a study using a defined ecosystem was needed to extrapolate the results. Significant decreases in defined community richness and diversity and changes in metabolic profile were observed after exposure to free chitosan. Free chitosan produced significant reductions in the abundance of the genera Lachnoclostridium, Anaerotignum, Blautia, Enterococcus, Eubacterium and Ruthenibacterium together with a slight decrease of the production of SCFAs, among other fermentation by-products. The immobilized chitosan significantly alleviated the impact caused by the antimicrobial polymer and significantly increased the relative abundance of the Bacteroidetes phylum compared to free chitosan. These results suggest the significance of assessing the impact of new ingredients and materials included in food on the human gut microbiota with models that simulate the gastrointestinal environment, such as in vitro bioreactor systems.

Efficient reduction in vegetative cells and spores of Bacillus subtilis by essential oil components-coated silica filtering materials.

Inactivation of bacterial spores is a key objective for developing novel food preservation technologies. In this work, the removal properties of filtering materials based on silica microparticles functionalized with essential oil components (EOCs) (carvacrol, eugenol, thymol, and vanillin) against Bacillus subtilis, a spore-forming bacterium, in two liquid matrices were investigated. The viability of vegetative cells and spores after treatment was also evaluated. The results exhibited marked removal effectiveness against B. subtilis vegetative cells and spores after filtration with the different silica supports coated with EOCs in either sterile water or nutrient broth, with reductions of 3.2 to 4.9 log units and 3.7 to 5.0 log units for vegetative cells and spores, respectively. The fluorescent viability images revealed the poor viability of the treated B. subtilis vegetative cells and spores due to damage to the cell envelope when coming into contact with the immobilized antimicrobials. The culture counts results revealed the great inhibitory capacity of the EOC-functionalized silica microparticles against B. subtilis vegetative cells and spores after a single filtration. Hence, the present work suggests the feasibility of using EOC-functionalized supports as filtering aids to enhance the microbial quality of liquid matrices with spore-forming microorganisms. PRACTICAL APPLICATION: The developed antimicrobial-coated filters have shown remarkable removal properties against an important spore-forming bacterium in food industry. These filters may be used as a potential sterilization technique for preservation of different beverages alone or in combination with other mild-thermal or nonthermal techniques.

Evaluation of the influence of food intake on the incorporation and excretion kinetics of mesoporous silica particles in C.elegans.

The effect of the presence of food on the incorporation and excretion of silica particles was studied in this work using the biological model Caenorhabditis elegans and image analysis techniques. The experiment was based on two 24-hour phases: exposure and depuration. During exposure, nematodes were maintained for 24 h in liquid medium with silica particles, but some with and others without food. During depuration, nematodes were transferred to medium without particles. Nematodes were analysed by an image analysis in both phases to quantify the properties of particle distributions in nematodes' bodies with time. No differences were found in the proportion of nematodes carrying particles in the exposure phase when food was present. However in the depuration phase, lack of food generated a high proportion of particle carriers. Particle distribution properties were also similar in the exposure phase. Nevertheless, lack of food produced particle accumulation due to decelerated excretion because digestive tube relaxed under these conditions. Thus after the depuration phase, lack of food led particles to persist in digestive tubes. According to these results, intake of silica particles had no retention effects when a food flux was provided, but particles were not easily excreted when the food flux was interrupted.

Comparative cytotoxic study of silica materials functionalised with essential oil components in HepG2 cells.

This work evaluated the cytotoxic effect of different EOCs-functionalised silica particle types. The in vitro toxicity of eugenol and vanillin-immobilised SAS, MCM-41 microparticles and MCM-41 nanoparticles was evaluated on HepG2 cells, and compared to free EOCs and pristine materials. The results revealed that free essential oil components and bare silica had a mild cytotoxic effect on HepG2 cells. However, the comparative study showed that free eugenol and vanillin had a milder cytotoxic effect than the equivalent concentrations of immobilised components on the different silica particles, while differences in cell viability between the bare and functionalised particles relied on the type of analysed material. The most cytotoxic materials were eugenol and vanillin-functionalised MCM-41 micro with IC50 values of 0.19 and 0.17 mg/mL, respectively, at 48 h exposure. Differences in cytotoxicity between functionalised particles may be attributed to the density of the functional components on their surface as a result of the functionalisation reaction performance for different materials. The study of the physico-chemical properties of particles demonstrated that cationic nature and increased hydrophobicity could be responsible for promoting cell-particle interactions for the eugenol and vanillin functionalised silica particles, enhancing their cytotoxic behaviour.

Degradation of silica particles functionalised with essential oil components under simulated physiological conditions.

In this work, the biodurability of three silica particle types (synthetic amourphous silica, MCM-41 microparticles, MCM-41 nanoparticles) functionalised with three different essential oil components (carvacrol, eugenol, vanillin) was studied under conditions that represented the human gastrointestinal tract and lysosomal fluid. The effect of particle type, surface immobilised component and mass quantity on the physico-chemical properties of particles and silicon dissolution was determined. Exposure to biological fluids did not bring about changes in the zeta potential values or particle size distribution of the bare or functionalised materials, but the in vitro digestion process partially degraded the structure of the MCM-41 nanoparticles. Functionalisation preserved the structure of the MCM-41 nanoparticles after simulating an in vitro digestion process, and significantly decreased the amount of silicon dissolved after exposing different particles to both physiological conditions, independently of the essential oil component anchored to their surface. The MCM-41 microparticles showed the highest solubility, while synthetic amorphous silica presented the lowest levels of dissolved silicon. The study of these modified silica particles under physiological conditions could help to predict the toxicological behaviour of these new materials.

Toxicological implications of amplifying the antibacterial activity of gallic acid by immobilisation on silica particles: A study on C. elegans.

Immobilisation of natural compounds on solid supports to amplify antimicrobial properties has reported successful results, but modifications to physico-chemical properties can also imply modifications from a toxicological viewpoint. This work aimed to study the immobilising process of gallic acid in the antibacterial activity of L. innocua and its toxicological properties in vivo using Caenorhabditis elegans. The experiment was based on obtaining the minimum bactericidal concentration for free and immobilised gallic acid by comparing lethality, locomotion behaviour, chemotaxis and thermal stress resistance on C.elegans at those concentrations. The results showed a lowering minimum bactericidal concentration and modifications to nematode responses. Increased lethality and velocity of movements was observed. Immobilisation increased the repellent effect of gallic acid with a negative chemotaxis index. Thermal stress resistance was also affected, with higher mortality for immobilised gallic acid compared to bare particles and free gallic acid. Thus despite evidencing a generalised increase in the toxicity of gallic acid in vivo, lowering the minimum bactericidal concentration allowed a bacterial reduction of 99 % with less than one third of mortality for the nematodes exposed to free gallic acid.

[Encapsulation and controlled delivery systems based on molecular gates]. / Sistemas de encapsulación y liberación controlada basados en el uso de puertas moleculares.

Mesoporous silica particles (MSP) are structures of silicon dioxide arranged so that they are able to create pores between 2 and 50 nm. The high volume of pores and the internal surface of the MSP make them excellent supports for the encapsulation of bioactive molecules. In addition, the possibility of including molecules acting as molecular gate onto their outer surface allows the design of smart delivery systems. Gated-MSP show "zero release" of the encapsulated molecule, but after the application of a specific external stimulus, the cargo is released as a specific response to the stimulus. This article describes the features of the MSP used in the encapsulation of bioactive compounds, the most important molecular gates to create controlled release systems, as well as examples of application of MSP for the encapsulation and controlled release of food ingredients and nutraceuticals. These applications include the modulation of the bioaccessibility of food ingredients or nutraceuticals as well as the protection of their stability against external agents degradation.

Las partículas mesoporosas de sílice (PMS) son estructuras de dióxido de silicio organizadas de manera que se crean poros entre 2 y 50 nm. El alto volumen de poros y su superficie interna, convierten a las PMS en excelentes soportes para la encapsulación de moléculas bioactivas. Además, la posibilidad de incluir moléculas con función de puerta molecular en su superficie externa permite el diseño de sistemas inteligentes de liberación. Las PMS con puerta molecular muestran "liberación cero" de la molécula encapsulada, pero tras la aplicación de un estímulo externo específico son capaces de liberar su carga como respuesta específica a dicho estímulo.En este artículo se describen las características de las PMS usadas en la encapsulación de compuestos bioactivos, las puertas moleculares más importantes para crear sistemas de liberación controlada y ejemplos de aplicación de PMS para la encapsulación de ingredientes alimenticios y nutracéuticos. Estas aplicaciones incluyen la modulación de la bioaccesibilidad de ingredientes alimenticios o nutracéuticos, así como la protección de su estabilidad frente a la degradación por agentes externos.

Improving the Antimicrobial Power of Low-Effective Antimicrobial Molecules Through Nanotechnology.

The objective of this work was on the one hand to assess the antibacterial activity of amines anchored to the external surface of mesoporous silica particles against Listeria monocytogenes in comparison with the same dose of free amines as well. It was also our aim to elucidate the mechanism of action of the new antimicrobial device. The suitability of silica nanoparticles to anchor, concentrate and improve the antimicrobial power of polyamines against L. monocytogenes has been demonstrated in a saline solution and in a food matrix. Moreover, through microscope observations it has been possible to determine that the attractive binding forces between the positive amine corona on the surface of nanoparticles and the negatively charged bacteria membrane provoke a disruption of the cell membrane. The surface concentration of amines on the surface of the nanoparticles is so effective that immobilized-amines were 100 times more effective in killing L. monocytogenes bacteria than the same amount of free polyamines. This novel approach for the creation of antimicrobial nanodevices opens the possibility to put in value the antimicrobial power of natural molecules that have been discarded because of its low antimicrobial power. PRACTICAL APPLICATION: Consumers demand for high-quality products, free from chemical preservatives, with an extended shelf-life. In this study, a really powerful antimicrobial agent based on a nanomaterial functionalized with a non-antimicrobial organic molecule was developed as a proof of concept. Following this approach it could be possible to develop a new generation of natural and removable antimicrobials based on their anchoring to functional surfaces for food, agricultural or medical purposes.

Anchoring Gated Mesoporous Silica Particles to Ethylene Vinyl Alcohol Films for Smart Packaging Applications.

This work is a proof of concept for the design of active packaging materials based on the anchorage of gated mesoporous silica particles with a pH triggering mechanism to a packaging film surface. Mesoporous silica micro- and nanoparticles were loaded with rhodamine B and functionalized with N-(3-trimethoxysilylpropyl)diethylenetriamine. This simple system allows regulation of cargo delivery as a function of the pH of the environment. In parallel, poly(ethylene-co-vinyl alcohol) films, EVOH 32 and EVOH 44, were ultraviolet (UV) irradiated to convert hydroxyl moieties of the polymer chains into ⁻COOH functional groups. The highest COOH surface concentration was obtained for EVOH 32 after 15 min of UV irradiation. Anchoring of the gated mesoporous particles to the films was carried out successfully at pH 3 and pH 5. Mesoporous particles were distributed homogeneously throughout the film surface and in greater concentration for the EVOH 32 films. Films with the anchored particles were exposed to two liquid media simulating acidic food and neutral food. The films released the cargo at neutral pH but kept the dye locked at acidic pH. The best results were obtained for EVOH 32 irradiated for 15 min, treated for particle attachment at pH 3, and with mesoporous silica nanoparticles. This opens the possibility of designing active materials loaded with antimicrobials, antioxidants, or aromatic compounds, which are released when the pH of the product approaches neutrality, as occurs, for instance, with the release of biogenic amines from fresh food products.

Antimicrobial activity of commercial calcium phosphate based materials functionalized with vanillin.

Infections represent one of the most frequent causes of arthroplasty revision. Thus, design of new antimicrobial scaffolds to reduce implant rejections, bone infections and associated medical costs is highly desired. In recent years, essential oil components (EOCs) have merged as compounds with significant antimicrobial activity that can be attached to specific surfaces to enhance and prolong their antimicrobial effect. Herein calcium phosphate CaP regenerative materials have been coated with a vanillin derivative to combine its original bone regeneration properties with antimicrobial action of EOCs. Materials in form of microparticles and blocks were prepared and fully characterized. Clonogenic viability tests demonstrated that low concentrations of material (10 mg·mL-1) resulted effective to kill 100% of E. coli DH5α bacteria. Additionally, vanillin containing scaffolds did not display any toxic effect over cells, yet they preserve the ability to express alkaline phosphatase (ALPL), collagen type 1, chain α1 (COL1A1) and bone gamma-carboxyglutamic acid-containing protein or osteocalcin (BGLAP), which are genes typically expressed by osteoblasts. These results demonstrate that commercially available scaffolds can be functionalized with EOCs, achieving antimicrobial activity and open up a new approach for the treatment and prevention of infection. STATEMENT OF SIGNIFICANCE: During the last years, the interest in bone regenerative materials with antibiotic properties has increased, since prosthesis infection is one of the most usual complications in implant surgery. In this work, we report a hybrid system composed by a calcium phosphate material (powders and scaffolds) functionalized with the derivative of an essential oil component (EOC). Our purpose was to provide the calcium phosphate material with antimicrobial activity without harming its bone regenerative capability. The obtained results were encouraging, which opens up the possibility of developing new modified materials for the prevention and treatment of bone infection.

Enhanced antimicrobial activity of essential oil components immobilized on silica particles.

The antimicrobial activity of essential oils components (EOCs) is well-known. However, their high volatility and powerful aroma limit their application in the formulation of a wide range of food products. In this context, the antimicrobial activity of carvacrol, eugenol, thymol and vanillin grafted onto the surface of three silica supports with different morphologies, textural properties and chemical reactivities (fumed silica, amorphous silica and MCM-41) was evaluated herein. Materials characterization revealed a good immobilization yield and all the devices showed a micro-scale particle size. Sensory evaluation revealed that sensory perception of EOCs decreases after covalent immobilization. Moreover, immobilization greatly enhanced the antimicrobial activity of the essential oil components against Listeria innocua and Escherichia coli compared to free components. The incorporation of EOCs immobilized on silica particles into pasteurized milk inoculated with L. innocua demonstrated their effectiveness not only for in vitro conditions, but also in a real food system.

Protection of folic acid through encapsulation in mesoporous silica particles included in fruit juices.

Folic acid (FA) is a synthetic vitamin commonly used for food fortification. However, its vulnerability to processing and storage implies loss of efficiency, which would induce over-fortification by processors to obtain a minimum dose upon consumption. Recent studies have indicated potential adverse effects of FA overdoses, and FA protection during processing and storage could lead to more accurate fortification. In addition, sustained vitamin release after consumption would help improve its metabolism. The objective of this work was to study controlled FA delivery and stability in fruit juices to reduce potential over-fortification risks by using gated mesoporous silica particles (MSPs). The obtained results indicated that FA encapsulation in MSPs significantly improved its stability and contributed to controlled release after consumption by modifying vitamin bioaccessibility. These results confirmed the suitability of MSPs as support for controlled release and protection of bioactive molecules in food matrices in different food production and storage stages.

Encapsulation of folic acid in different silica porous supports: A comparative study.

Although folic acid is essential to numerous bodily functions, recent research indicates that a massive exposition to the vitamin could be a double-edged sword. In this study, the capacity of different caped mesoporous silica particles (i.e. Hollow Silica Shells, MCM-41, SBA-15 and UVM-7) to dose FA during its passage through the gastrointestinal tract has been evaluated. Results confirmed that the four capped materials were capable to hinder the delivery of FA at low pH (i.e. stomach) as well as able to deliver great amounts of the vitamin at neutral pH (i.e. intestine). Nevertheless, the encapsulation efficiency and the deliver kinetics differed among supports. While supports with large pore entrance exhibited an initial fast release, MCM-41, showed a sustained release along the time. This correlation between textural properties and release kinetics for each of the supports reveals the importance of a proper support selection as a strategy to control the delivery of active molecules.

Protective effect of mesoporous silica particles on encapsulated folates.

Mesoporous silica particles (MSPs) are considered suitable supports to design gated materials for the encapsulation of bioactive molecules. Folates are essential micronutrients which are sensitive to external agents that provoke nutritional deficiencies. Folates encapsulation in MSPs to prevent degradation and to allow their controlled delivery is a promising strategy. Nevertheless, no information exists about the protective effect of MSPs encapsulation to prevent their degradation. In this work, 5-formyltetrahydrofolate (FO) and folic acid (FA) were entrapped in MSPs functionalized with polyamines, which acted as pH-dependent molecular gates. The stability of free and entrapped vitamins after acidic pH, high temperature and light exposure was studied. The results showed the degradation of FO after high temperature and acidic pH, whereas entrapped FO displayed enhanced stability. Free FA was degraded by light, but MSPs stabilized the vitamin. The obtained results point toward the potential use of MSPs as candidates to enhance stability and to improve the bioavailability of functional biomolecules.